Cannabis sativa (Cannabis) is a multipurpose plant species consisting of specific lineages that for centuries has either been artificially selected for the production of fiber or the psychoactive drug Δ9-tetrahydrocannabinol (THC). With the recent lifting of previous legal restrictions on consuming Cannabis, there has been a resurgence of interest in understanding and manipulating Cannabis genetics to enhance its compositions. Yet, recently developed approaches are not amenable to high-throughput gene stacking to study multi-genic traits. Here, we demonstrate an efficient nanoparticle-based transient gene transformation protocol where multiple gene plasmids can be expressed simultaneously in intact Cannabis leaf cells in a very short time (5 days). Constructs encoding two soybean transcription factors were co-grafted onto poly-ethylenimine cationic polymer-modified silicon dioxide-coated gold nanoparticles (PEI-Au@SiO2). Infiltration of the DNA-PEI-Au@SiO2 into Cannabis leaf tissues resulted in the transcription of both soybean genes and the localization of fluorescent-tagged transcription factor proteins in the nuclei of Cannabis leaf cells including the trichomes, which are the cell types that biosynthesize valuable cannabinoid and terpene metabolites. Our study exemplifies a rapid transient gene transformation approach that will be useful to study the effects of gene stacking in Cannabis.

A common approach for analyzing large-scale molecular data is to cluster objects sharing similar characteristics. This assumes that genes with highly similar expression profiles are likely participating in a common molecular process. Biological systems are extremely complex and challenging to understand, with proteins having multiple functions that sometimes need to be activated or expressed in a time-dependent manner. Thus, the strategies applied for clustering of these molecules into groups are of key importance for translation of data to biologically interpretable findings. Here we implemented a multi-assignment clustering (MAsC) approach that allows molecules to be assigned to multiple clusters, rather than single ones as in commonly used clustering techniques. When applied to high-throughput transcriptomics data, MAsC increased power of the downstream pathway analysis and allowed identification of pathways with high biological relevance to the experimental setting and the biological systems studied. Multi-assignment clustering also reduced noise in the clustering partition by excluding genes with a low correlation to all of the resulting clusters. Together, these findings suggest that our methodology facilitates translation of large-scale molecular data into biological knowledge. The method is made available as an R package on GitLab (https://gitlab.com/wolftower/masc).

Protein normalization of western blots has relied upon housekeeping proteins which exhibit signal saturation and varied cellular expression level variations. These issues can produce spurious results leading to erroneous conclusions. A superior method to protein normalization using housekeeping proteins is Total Protein Normalization, a method now recognized as the gold standard for quantitative westerns. Total Protein Normalization requires that all proteins on a membrane be stained or labeled uniformly, imaged, and then analyzed for total protein. It is important that such a normalization process not interfere with typical immunodetection methods, fits within existing western workflows, and exhibits a linear relationship of signal intensity to protein load under all experimental conditions. Here we report that we developed a new reagent enabling Total Protein Normalization, and we demonstrate its superior protein normalization capabilities through analysis of target proteins in different cell backgrounds. These data illustrate how housekeeping proteins exhibit signal saturation, yield erroneous normalization data, and display sample-to-sample variations averaging 48.2 % overall. Signal intensities obtained using our new method show a linear relationship to protein sample load, thus providing accurate protein normalization with an overall average variation of 7.7 %.

cis-1,2-Dihydro-1,2-naphthalenediol (DHND) is a valuable molecule employed for the pharmaceutical synthesis of bioactive compounds, such as bicyclic conduritol analogues. Enantiopure (+)-(1R,2S)-DHND (>98 % ee) is easily biosynthesized through the dearomatizing dihydroxylation of naphthalene, catalyzed by toluene dioxygenase (TDO) from Pseudomonas putida F1. However, the opposite enantiomer (-)-(1S,2R)-DHND could not be directly accessed, neither by chemical synthesis nor via biocatalytic approaches. Herein, we report a one-step biosynthesis of the opposite enantiomer (-)-(1S,2R)-DHND in a recombinant TDO E. coli BW25113 platform. We based on a semi-rational approach to generate a set of TDO variants, targeting exclusively the hotspot position F366, in order to enable an enantiomeric switch in the generated product. Eight out of nine single point variants were active and showed not only an alteration in enantioselectivity, but also generated an enantiomeric excess of the pursued product. Variant TDOF366V outperformed above the rest of the set, enabling the synthesis of (-)-(1S,2R)-DHND not only with an excellent enantiomeric excess of 90 %, but also with an advantageous product formation. A comparative semi-preparative biosynthesis yielded, 287 mg of (+)-(1R,2S)-DHND (>98 % ee) and 101 mg of (-)-(1S,2R)-DHND (90 % ee), when performed in a total volume of 100 mL with TDO wild-type and TDOF366V resting cells, respectively.

Recombinant forms of the spike protein of SARS-CoV-2 and related viruses have proven difficult to produce with good yields in mammalian cells. Given the panoply of potential COVID-19 diagnostic tools and therapeutic candidates that require purified spike protein and its importance for ongoing SARS-CoV-2 research, we have explored new approaches for spike production and purification. Three transient gene expression methods based on PEI-mediated transfection of CHO or HEK293 cells in suspension culture in chemically-defined media were compared for rapid production of full-length SARS-CoV-2 spike ectodomain. A high-cell-density protocol using DXB11-derived CHOBRI/55E1 cells gave substantially better yields than the other methods. Different forms of the spike ectodomain were expressed, including the wild-type SARS-CoV-2 sequence and a mutated form (to favor expression of the full-length spike ectodomain stabilized in pre-fusion conformation), with and without fusion to putative trimerization domains. An efficient two-step affinity purification method was also developed. Ultimately, we have been able to produce highly homogenous preparations of full-length spike, both monomeric and trimeric, with yields of 100-150 mg/L in the harvested medium. The speed and productivity of this method support further development of CHO-based approaches for recombinant spike protein manufacturing.

The optimal conditions for high biomass and lipid production from Nannochloris atomus were evaluated. The parameters used in this study were light emitting diode (LED) wavelength mixing ratio, the photoperiod, salinity tolerance, and single and combined stresses. Biomass production was monitored in the first phase using red LED (625 nm), followed by lipid production by green LED (520 nm) in the second phase. The optimal conditions were obtained using a single red LED with light:dark durations of 20:4 h and two days of exposure in combined stresses of 1.06 M NaCl and green LED. Under these conditions, 68.6 % (w/w) lipid content were obtained. Compared to the non-stress control, the lipid content was increased by 31.9 %. Linolenic acid (C18:3) the omega-3 fatty acid was produced up to 52.4 % in 1.06 M NaCl as a single stress.

Lentiviral vectors have proven their great potential to serve as a DNA delivery tool for gene modified cell therapy and gene therapy applications. The downstream processing of these vectors is however still a great challenge, particularly because of the low stability of the virus. Harvesting and clarification are critical and until now insufficiently characterized steps for lentivirus processing. To address this bottleneck, we analyzed whether lentiviral vectors produced by transient transfection of HEK293 T/17 SF suspension cells can be efficiently clarified with a lab-scale method with the filter aid diatomaceous earth (DE) and bioburden reducing membrane filters achieving high lentivirus recoveries. Using a design of experiment approach we found that higher DE concentrations are advantageous for a higher turbidity reduction and shorter filtration times, but at the same time LV titer decreases with increasing DE concentration. A DE concentration of 9 g/L was identified with a DoE as a robust set-point. Clarification with DE was compared with for lab-scale traditionally employed centrifugation and subsequent bioburden reduction filtration of viral vectors. The use of DE allows to perform a harvest and clarification process, which does not only facilitate faster and safer virus handling, but enables a lower material consumption due to the extremely increased filter capacity, thus representing an efficient and robust lab-scale clarification process.

Obtaining large-scale hairy root cultures is a major challenge to increasing root biomass and secondary metabolite production. Enhanced production of stilbene compounds such as trans-resveratrol, trans-arachidin-1 and trans-arachidin-3 was achieved using an elicitor treatment procedure. Two different hairy root inoculum densities were investigated and compared between shake flask and bioreactor cultures. The lowest growth index was observed using a 20 g/L inoculum size in the bioreactor, which differed significantly from bioreactor of 5 g/L. Increasing the hairy root inoculum size from 5 g/L to 20 g/L in both the shake flask and bioreactor significantly improve antioxidant activity, phenolic content and stilbene compound levels. The highest ABTS and FRAP antioxidant activity, and levels of total phenolic compounds, trans-arachidin-1 and trans-arachidin-3 in the crude extract were demonstrated in shake flask cultures with a 20 g/L inoculum after elicitation for 72 h. The minimum inhibitory concentrations (MICs) of the crude extract to inhibit growth of foodborne microbes, S. aureus, S. typhimurium and E. coli, were 187.5, 250 and 500 μg/mL, respectively. This was due to the ability of the crude extract to disrupt the cell membrane, as observed by scanning electron microscopy (SEM) showing ruptured pores on the S. aureus and S. typhimurium cell surfaces. Moreover, the E. coli cell division process could be inhibited by the crude extract, which promoted an increase in cell size. A DNA nicking assay indicated that a 50 μg/mL concentration of the crude extract caused plasmid DNA damage that might be due to a genotoxic effect of the pro-oxidant activity of the crude extract.

In recent years, researches on selenium nanoparticle have gained more attention due to its important role in many physiological processes. Generally, selenium nanoparticle has a high level of absorption in regular supplementation comparative to selenium. Therefore it is all-important to develop new techniques to elevate the transportation of selenium compounds (selenoproteins, selenoenzymes, etc.) by increasing their bioavailability, bioactivity, and controlled release. SeNPs have special attention regarding their application as food additives and therapeutic agents. Selenium nanoparticle has biomedical and pharmaceutical uses due to its antioxidant, antimicrobial, antidiabetic, and anticancer effects. Selenium nanoparticle is also used to antagonize the toxic effect of chemical and heavy metals. SeNPs are beneficial for the treatment of water and soil contaminated with metals and heavy metals as it has adsorption capability. Selenium nanoparticle is synthesized by the bioreduction of selenium species (sodium selenate, sodium selenite, selenium dioxide, and selenium tetrachloride, etc.) by using bacteria, fungi, plant, and plant extracts, which have given hope for the bioremediation of selenium contaminated water and soils. This article reviews the procedure of selenium nanoparticle synthesis (physical, chemical and biological methods), characterization (UV-vis spectroscopy, transmission electron microscopy, Scanning electron microscopy, electron dispersive X-ray spectroscopy, X-ray diffraction, Fourier transform-infrared spectroscopy, etc.), with the emphasis on its role and application in health and environment.

Esters from branched alcohols and dicarboxylic linear acids are widely used as lube bases due to their good performance at low temperatures. This work proposes a new process to synthesize bis(2-ethylbutyl) adipate and bis(2-ethylbutyl) sebacate by using the lipase-based catalyst Novozym® 435 in a solvent-free system. Different reaction strategies have been tested in order to minimize 2-ethyl-1-butanol losses due to its evaporation and optimum operation conditions have been determined: 2.5 % of biocatalyst, 50 °C and a molar excess of alcohol of 15 % for the adipic diester and of 25 % for the sebacic one. It has also been proven that the immobilized enzyme can be reused in seven successive reaction cycles, achieving high yields without an appreciable reduction of activity. This biocatalytic pathway is a promising basis for the development of a more sustainable large scale process for obtaining biodegradable lubricants, as it is pointed out by productivity, economic and green metrics calculations.

Eggshell membrane, an eco-compatible, safe and cheap by-product was employed as carrier for the laccase from Trametes versicolor immobilization. In order to evaluate the best protocol to apply for the syringic acid degradation, two different types of laccase loading on eggshell membrane were used by incubation in solution or by enzyme-dropping. Chemicals (covalent) and physicals (adsorption) immobilizations were tested for both procedure using native or periodate-oxidized laccase. It is shown that immobilization of periodate-oxidized laccase on NiCl2-pretreated eggshell membrane was the best method for the first procedure (immobilized activity 1300 U/Kg, a residual activity of 30 % for 6 reuse). For the enzyme-dropping protocol a covalent method with the bifunctional cross linker (glutaraldehyde) was the best method (immobilized activity 3500 U/Kg, a residual activity of 45 % for 6 reuse).

Deracemization of D,L-phosphinothricin (D,L-PPT) is one of the most promising routes for preparation of optically pure L-PPT. In this work, an efficient multi-enzyme redox cascade was developed for deracemization ofPPT, which includes oxidative reaction and reductive reaction. The oxidative reaction catalyzing oxidative deamination of D-PPT to 2-oxo-4-[(hydroxy)(-methyl)phosphinyl]butyric acid (PPO) was performed by a D-amino acid oxidase and a catalase for removing H2O2. The reductive reaction catalyzing amination of PPO to L-PPT is achieved by a glufosinate dehydrogenase and a glucose dehydrogenase for cofactor regeneration. To avoid the inhibitory effect of glucose on the oxidative reaction, a "two stages in one-pot" strategy was developed to combine these two reactions in deracemization process. By using this strategy, the L-PPT was obtained with a high yield (89 %) and > 99 % enantiomeric excess at substrate loading of 300 mM in absence of addition of extra NADP+. These encouraging results demonstrated that the developed enzyme cascade deracemization process exhibits great potential and economical competitiveness for manufacture of L-PPT from D,L-PPT.

To reduce dependence on petroleum, the biosynthesis of important chemicals from simple substrates using industrial microorganisms has attracted increased attention. Metabolic engineering of Saccharomyces cerevisiae offers a sustainable and flexible alternative for the production of various chemicals. As a key metabolic intermediate, malonyl-CoA is a precursor for many useful compounds. However, the productivity of malonyl-CoA derivatives is restricted by the low cellular level of malonyl-CoA and enzymatic performance. In this review, we focused on how to increase the intracellular malonyl-CoA level and summarize the recent advances in different metabolic engineering strategies for directing intracellular malonyl-CoA to the desired malonyl-CoA derivatives, including strengthening the malonyl-CoA supply, reducing malonyl-CoA consumption, and precisely controlling the intracellular malonyl-CoA level. These strategies provided new insights for further improving the synthesis of malonyl-CoA derivatives in microorganisms.

The asymmetric reduction of ketones is a frequently used synthesis route towards chiral alcohols. Amongst available chemo- and biocatalysts the latter stand out in terms of product enantiopurity. Their application is, however, restricted by low reaction output, often rooted in limited enzyme stability under operational conditions. Here, addition of 2-hydroxypropyl-β-cyclodextrin to bioreductions of o-chloroacetophenone enabled product concentrations of up to 29 % w/v at full conversion and 99.97 % e.e. The catalyst was an E. coli strain co-expressing NADH-dependent Candida tenuis xylose reductase and a yeast formate dehydrogenase for coenzyme recycling. Analysis of the lyophilized biocatalyst showed that E. coli cells were leaky with catalytic activity found as free-floating enzymes and associated with the biomass. The biocatalyst was stabilized and activated in the reaction mixture by 2-hydroxypropyl-β-cyclodextrin. Substitution of the wild-type xylose reductase by a D51A mutant further improved bioreductions. In previous optimization strategies, hexane was added as second phase to protect the labile catalyst from adverse effects of hydrophobic substrate and product. The addition of 2-hydroxypropyl-β-cyclodextrin (11 % w/v) instead of hexane (20 % v/v) increased the yield on biocatalyst 6.3-fold. A literature survey suggests that bioreduction enhancement by addition of cyclodextrins is not restricted to specific enzyme classes, catalyst forms or substrates.

Small molecules inhibitors of neuraminidases (NAs) are ones of the most prospective molecules proposed for the treatment of influenza viruses. The determination of their inhibition activity in vitro is an important step during the development of antiviral drugs. However, the analytical methods typically used for the evaluation of NA activity and inhibition (fluorescence-based assays using MUNANA substrate or thiobarbituric acid assay, TBA) may suffer from interferences caused by tested inhibitors as signal quenching or self-fluorescence, moreover in TBA are used toxic and carcinogenic reagents. The determination of the NA activity can be effectively performed by alternative methods based on lectin - glycan recognition, usually as enzyme-linked lectin assay (ELLA). We have adapted the ELLA assay to a lectin-based assay in a microplate format with fluorescence detection for determination of NA inhibitory activity. We optimized our protocol and the developed method was tested using four different small molecule NA inhibitors or potential NA inhibitors (DANA, zanamivir, quercetin and α-mangostin) with three bacterial NAs (from Clostridium perfringens, Vibrio cholerae and Arthrobacter ureafaciens), and the IC50 values for NA inhibitors were determined. The inhibition effect of DANA was observed for all 3 tested NAs (IC50 = 10.1 μM for V. cholerae, 13.4 μM for C. perfringens and 402.9 μM for A. ureafaciens, respectively) and of Zanamivir only for NA from V. cholerae (IC50 = 101.9 μM). For both quercetin and α-mangostin, no inhibition effect to the tested NAs was observed. The main advantages of herein described method are good sensitivity due to fluorescent signal detection, the absence of the interference caused by fluorescent signal quenching by tested inhibitors, the use of natural substrates (glycoproteins) and the avoiding the use of toxic reagents.

Fe3O4 nanoparticles were obtained by chemical coprecipitation of iron chloride and sodium hydroxide. The morphology and sizes of the obtained nanoparticles were characterized using laser Doppler velocimetry, transmission electron and atomic force microscopy. Then the nanoparticles were stabilized by three polycations (polyethylenimine (PEI), poly(allylamine hydrochloride) (PAH), poly(diallyldimethylammonium chloride) (PDADMAC)) to increase their biocompatibility. The cytotoxicity of the obtained polymer-stabilized nanoparticles was studied using a human lung carcinoma cell line (A549). The biodistribution of nanoparticles stabilized by polycations in human lung carcinoma cells was analyzed by transmission electron microscopy, and the toxicity of nanomaterials was evaluated using toxicity tests and flow cytometry. As a result, the most biocompatible nanoparticle-biopolymer complex was identified. PAH stabilized magnetic nanoparticles demonstrated the best biocompatibility, and the PEI-magnetic nanoparticle complex was the most toxic.

Claviceps purpurea is a filamentous fungus well known as a widespread plant pathogen, but it is also an important ergot alkaloid producer exploited by the pharmaceutic industry. In this work, we demonstrated that CRISPR/Cas9 can be a tool for directed mutagenesis in C. purpurea targeting pyr4 and TrpE genes encoding the orotidine 5'-phosphate decarboxylase involved in pyrimidine biosynthesis and the α-subunit of the anthranilate synthase involved in tryptophan biosynthesis, respectively. After protoplast transformation and single spore isolation, homokaryotic mutants showing uridine or tryptophan auxotrophy were selected. In all cases, insertions or insertions combined with deletions were found mostly 3 bp upstream of the PAM sequence. However, transformation efficiencies of CRISPR/Cas9 and CRISPR/Cas9 mediated homology-directed repair only slightly improved in comparison to homologous recombination-mediated knocking-out of the TrpE gene. Moreover, Trp auxotrophs were non-infectious towards rye plants likely due to a decreased production of the plant hormones auxins, which are synthesized by C. purpurea from indole-3-glycerolphosphate in Trp-dependent and Trp-independent biosynthetic pathways, and help the fungus to colonize the plant host. It was demonstrated that the CRISPR/Cas9 vector containing autonomous replicative sequence AMA1 can be fully removed by further culturing of C. purpurea on non-selective media. This method enables introducing multiple mutations in Claviceps and makes feasible metabolic engineering of industrial strains.

Protein topology engineering has emerged as a new dimension to alter protein stability and function. Inspired by the art of nature, where backbone cyclization is frequently adopted to enhance the stability of natural peptide products and thermostable enzymes; herein, we report protein topology engineering of an industrial thermolabile gamma lactamase via catenation. Two different protein catenanes were successfully constructed via SpyTag/SpyCatcher modules and two different peptide dimer domains. The designed protein catenanes were functionally synthesized in Escherichia coli. A comparison of their biochemical properties revealed that protein topology played a key role in the stability of gamma lactamase. Protein catenation enhanced both the thermo- and proteolytic stabilities of gamma lactamase. Gamma lactamase was stabilized by ∼8 °C in one of the catenated forms. Moreover, Cat1-MhIHL-V54L and Cat2-MhIHL-V54L displayed 1.8- and 2.4-fold higher enzyme efficiencies (Kcat/Km), respectively, than the unattenuated enzyme. Therefore, our results proved that protein catenane construction could be a general strategy to strengthen industrial biocatalysts by mechanisms distinct from those of the conventional direct evolution schemes, whereby our results offer wide applications in the fine chemical industry.

Calcifediol (25(OH)VD3) is a physiologically very important vitamin D3 metabolite and of high pharmaceutical importance, due to its potential for treating not only vitamin D3 deficiencies but also coronary diseases and cancer. Previously, we established a whole-cell Bacillus megaterium-based system using the cytochrome P450 CYP109A2 for the biotransformation of vitamin D3 into its metabolite 25-hydroxyvitamin D3. In this study, we demonstrate the importance of the region between amino acids T103 and A106 for the catalytic activity of CYP109A2 towards vitamin D3 as a substrate. In order to increase the productivity of the system, reaction conditions (xylose, vitamin D3, saponin, 2-hydroxypropyl-β-cyclodextrin) were optimized for the in vivo production of 25-hydroxyvitamin D3. With cells producing the T103A mutant, a productivity of 282.7 mg/L/48 h was achieved under the optimized conditions. This value is two times higher than that obtained in the control reaction with the wild-type enzyme in this study and five times higher than that obtained in a previous study.

Efficient biotransformation of lignin requires the activity of different oxidative enzymes. In this work, 19 bacterial multi-copper oxidases were screened for oxidase activity against 19 soluble substrates and revealed the highest activity in the laccase CotABsu (BSU0630) from Bacillus subtilis. Structure-based site-directed mutagenesis of CotABsu identified four conserved residues (His419, Cys492, His497, and Met502) as critical for activity against 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS). Greatly reduced oxidase activity was found in the CotABsu mutant proteins E213A, N214A, C229A, N264A, E298A, T415A, R416A, Q468A, and T480A. We also designed a lignin-agarose plate screen for detecting oxidase activity of purified proteins against polymeric lignin, which confirmed the results obtained with ABTS and identified three mutant variants with increased activity toward kraft lignin (E213A, T415A, and T260A). X-ray photoelectron spectroscopy analysis of low sulfonate kraft lignin after incubation with CotABsu revealed a reduction in the content of CC/CC bonds and increase in CO/CO bonds. Product analyses using mass spectrometry, liquid chromatography, and bright-field microscopy revealed an increased polymerization state of reaction products suggesting that formation of radical intermediates was followed by radical coupling. Our results provide further insights into the mechanisms of lignin oxidation by laccases.

O-Succinyl-l-homoserine (OSH) is an important platform chemical in production of C4 chemicals such as succinic acid, homoserine lactone, γ‑butyrolactone, and 1,4‑butanediol. The production of OSH through chemical method or the current engineering strain is difficult and not optimal, and thereby there remains a need to develop new engineering strategy. Here, we engineered an OSH overproducing Escherichia coli strain through deleting the degradation and competitive pathways, overexpressing thrA and metL to enhance the metabolic flux from l-asparate to l-homoserine. Additionally, increasing the precursor succinyl-CoA supply through simultaneously knocking out sucD and overexpressing sucA further increased the yield of OSH. The engineered strain OSH9/pTrc-metA11-yjeH with above strategies produced OSH at the concentration of 24.1 g/L (0.609 g/g glucose) in batch fermentation. To gain detailed insight into metabolism of the engineered strain, comparative metabolic profiling was performed between the engineered and wide-type strain. The metabolomics data deciphered that the carbon was directed toward the OSH biosynthesis resulting in less flexibility of the genetically modified strain than the wide-type strain.

Oxygen supply implies higher production cost and reduction of maximum theoretical yields. Thus, generation of fermentation products is more cost-effective. Aiming to find a key piece for the production of (poly)-3-hydroxybutyrate (PHB) as a fermentation product, here we characterize an acetoacetyl-CoA reductase, isolated from a Candidatus Accumulibacter phosphatis-enriched mixed culture, showing a (kcatNADH/KMNADH)/(kcatNADPH/KMNADPH)>500. Further kinetic analyses indicate that, at physiological concentrations, this enzyme clearly prefers NADH, presenting the strongest NADH preference so far observed among the acetoacetyl-CoA reductases. Structural and kinetic analyses indicate that residues between E37 and P41 have an important role for the observed NADH preference. Moreover, an operon was assembled combining the phaCA genes from Cupriavidus necator and the gene encoding for this NADH-preferring acetoacetyl-CoA reductase. Escherichia coli cells expressing that assembled operon showed continuous accumulation of PHB under oxygen limiting conditions and PHB titer increased when decreasing the specific oxygen consumption rate. Taken together, these results show that it is possible to generate PHB as a fermentation product in E. coli, opening opportunities for further protein/metabolic engineering strategies envisioning a more efficient anaerobic production of PHB.

As the periodic emission of light pulses by light emitting diodes (LEDs) is known to stimulate growth or induce high value biocompounds in microalgae, this flashing light regime was tested on growth and biochemical composition of the microalgae Nannochloropsis gaditana, Koliella antarctica and Tetraselmis chui. At low flashing light frequencies (e.g., 5 and 50 Hz, Duty cycle = 0.05), a strain-dependent growth inhibition and an accumulation of protein, polyunsaturated fatty acids, chlorophyll or carotenoids (lutein, β-carotene, violaxanthin and neoxanthin) was observed. In addition, a 4-day application of low-frequency flashing light to concentrated cultures increased productivities of eicosapentaenoic acid (EPA) and specific carotenoids up to three-fold compared to continuous or high frequency flashing light (500 Hz, Duty cycle = 0.05). Therefore, applying low-frequency flashing light as finishing step in industrial production can increase protein, polyunsaturated fatty acids or pigment contents in biomass, leading to high-value algal products.

We describe here a vector construct to establish homogeneous cell populations expressing a recombinant gene of interest (GOI) at tuneable levels, including low expression levels that are difficult to generate using standard cell line development techniques. This is achieved using a tricistronic mRNA that contains an open reading frame for the gene of interest, a first internal ribosome entry site (IRES), an open reading frame for a fluorescent reporter protein (such as green fluorescent protein, GFP), a second IRES and an open reading for an antibiotic resistance gene (such as puromycin N-acetyl-transferase, PAC). The resistance gene allows convenient selection of stable cell populations. The fluorescent reporter protein allows convenient homogeneity and expression stability assessments of the cell line. The expression level of the GOI can be adjusted by using different start codons for the open reading frame. These alternate start codons will initiate the translation of the GOI with different efficiency, leading to cell populations expressing different levels of the GOI, and similar levels of the fluorescent reporter through the first IRES and the puromycin resistance gene through the second IRES to the GOI. Such cell populations are useful tools, for instance to assess the safety of potent targeted therapeutics, as they allow the simplified generations of homogenous cell populations with different levels of target protein expression between populations.

Activin A plays a central role in the differentiation of stem cells into definitive endoderm, the first step in embryonic development and function development in many organ systems. The aims of this study were to induce controlled and fine-tuned cell differentiation using a gradient nanotechnology and compare this with a classic protocol and to investigate how induced pluripotent stem cells differentiated depending on the gradual increase of Activin A. The density difference was tested by attaching Activin A to a gold nanoparticle gradient for high-precision density continuity. Cells expressed the definitive endoderm markers SRY-box transcription factor 17 and transcription factor GATA-4 to different extents along the gradient, indicating a density-dependent cell response to Activin A. In both the gradient and the classic differentiation setups, the protein expression increased from days 1 to 5, but a significant increase already on day 3 was found only in the gradient-based setup. By utilizing the gradient technology to present the right amount of active biomolecules to cells in vitro, we were able to find an optimal setting for differentiation into definitive endoderm. The use of gradient surfaces for differentiation allows for improvements, such as efficiency and faster differentiation, compared with a classic protocol.

In this study, we investigated oxidative stress in the green microalgae, Chlorella sorokiniana, in co-culture with the plant growth promoting bacteria (PGPB), Azospirillum brasilense. This relationship was studied in the absence of an exogenous stressor, under copper stress, and under nitrogen limitation stress. We confirmed that copper and nitrogen limitation induced algal oxidative stress and reductions in chlorophyll content. In all cases, the presence of A. brasilense lowered the accumulation of intracellular reactive oxygen species (ROS) while promoting chlorophyll content. This effect was driven, in part, by A. brasilense's secretion of the auxin hormone, indole-3-acetic acid, which is known to mitigate stress in higher plants. The findings of the present study show that stress mitigation by A. brasilense resulted in suppressed starch accumulation under nitrogen limitation stress and neutral lipid accumulation under copper stress. In fact, A. brasilense could almost completely mitigate oxidative stress in C. sorokiniana resulting from nitrogen limitation, with ROS accumulation rates comparable to the axenic control cultures. The biotechnological implication of these findings is that co-culture strategies with A. brasilense (and similar PGPB) are most effective for high growth applications. A second growth stage may be needed to induce accumulation of desired products.

The fermentative production of biobased chemicals and polymers using crude lignocellulose hydrolysates is challenging due to the presence of various inhibitory compounds and multiple sugars. This study evaluates the metabolic response of Actinobacillus succinogenes for the production of succinic acid using spent sulphite liquor (SSL) as feedstock derived from industrial acidic sulphite pulping of Eucalyptus globulus hardwood. A transcriptomic approach led to significant insights on gene regulation of the major metabolic pathways (glycolysis, pentose phosphate pathway, TCA cycle, pyruvate metabolism and oxidative phosphorylation) in batch cultures carried out on SSL and compared with glucose and xylose. Significantly overexpressed genes in SSL compared to glucose and xylose were fructose biphosphate aldolase (> 1.18-fold change) in the catabolism, phosphoenolpyruvate carboxykinase (> 1.59-fold change) and malate dehydrogenase (> 1.49-fold change) in the TCA cycle, citrate lyase (> 1.7-fold change), dihydrolipoamide dehydrogenase (> 0.88-fold change), pyruvate dehydrogenase E2 (> 1.63-fold change) and pyruvate formate lyase (> 0.61-fold change), involved in acetyl-CoA pathways. Finally, C4 tricarboxylic transporters were overexpressed (DCU (> 1.61-fold change) and 0079 (> 4.19-fold change). SSL was responsible for the upregulation of genes involved in the TCA cycle and oxidative phosphorylation, while xylose showed similar results with SSL in the oxidative phosphorylation.

Respiratory syncytial virus (RSV) is a highly contagious virus causing severe infection in infants and the elderly. Various approaches are being used to develop an effective RSV vaccine. The RSV fusion (F) subunit, particularly the cleaved trimeric pre-fusion F, is one of the most promising vaccine candidates under development. The pre-fusion conformation elicits the majority of neutralizing antibodies during natural infection. However, this pre-fusion conformation is metastable and prone to conversion to a post-fusion conformation, thus hindering the potential of this construct as a vaccine antigen. The Vaccine Research Center (VRC) at the National Institutes of Health (NIH) designed a structurally stabilized pre-fusion F glycoprotein, DS-Cav1, that showed high immunogenicity and induced a neutralizing response in animal studies. To advance this candidate to clinical manufacturing, a production process that maintained product quality (i.e. a cleaved trimer with pre-fusion conformation) and delivered high protein expression levels was required. This report describes the development of the vaccine candidate including vector design and cell culture process development to meet these challenges. Co-transfection of individual plasmids to express DS-Cav1 and furin (for DS-Cav1 cleavage and activation) demonstrated a superior protein product expression and pre-fusion conformation compared to co-expression with a double gene vector. A top clone was selected based on these measurements. Protein expression levels were further increased by seeding density optimization and a biphasic hypothermia temperature downshift. The combined efforts led to a high-yield fed-batch production of approximately 1,500 mg/L (or up to 15,000 doses per liter) at harvest. The process was scaled up and demonstrated to be reproducible at 50 L-scale for toxicity and Phase I clinical trial use. Preliminary phase I data indicate the pre-fusion antigen has a promising efficacy (Crank et al., 2019).

Complete submergence (Sub) imposes detrimental effects on growth and survival of crop plants, including rice. Here, we investigated the beneficial effects of reduced glutathione (GSH) in mitigating Sub-induced adverse effects in two high-yielding rice cultivars BRRI dhan29 and dhan52. Both cultivars experienced growth defects, severe yellowing, necrosis and chlorosis, when they were completely immersed in water for 14 days. The poor growth performance of these cultivars was linked to biomass reduction, decreased levels of photosynthetic pigments and proline, increased levels of H2O2 and malondialdehyde, and declined activities of enzymatic antioxidants like superoxide dismutase, ascorbate peroxidase, peroxidase, catalase, glutathione peroxidase and glutathione S-transferase. Pretreatment with exogenous GSH led to significant growth restoration in both cultivars exposed to Sub. The elevated Sub-tolerance promoted by GSH could partly be attributed to increased levels of chlorophylls, carotenoids, soluble proteins and proline. Exogenous GSH also mitigated Sub-induced oxidative damage, as evidenced from reduced levels of H2O2 and malondialdehyde in accordance with the increased activities of antioxidant enzymes. Results revealed that dhan52 was more tolerant to Sub-stress than dhan29, and GSH successfully rescued both cultivars from the damage of Sub-stress. Collectively, our findings provided an insight into the GSH-mediated active recovery of rice from Sub-stress, thereby suggesting that external supply of GSH may be an effective strategy to mitigate the adverse effects of Sub in rice.

Flavonoids have notable biological activities and have been widely used in the medicinal and chemical industries. However, single-copy integration of heterologous pathway genes limits the production of flavonoids. In this work, we designed and constructed single-step integration of multiple flavonoid (2S)-naringenin biosynthetic pathway genes in S. cerevisiae. The efficiency of the naringenin metabolic pathway gene integration into the rDNA site reached 93.7%. Subsequently, we used a high titer p-coumaric acid strain as a chassis, which eliminated feedback inhibition of tyrosine and downregulated the competitive pathway. The results indicated that increasing the supply of p-coumaric acid was effective for naringenin production. We additionally optimized the amount of donor DNA. The optimum strain produced 149.8 mg/L of (2S)-naringenin. The multi-copy integration of flavonoid pathway genes effectively improved (2S)-naringenin production in S. cerevisiae. We further analyzed the copy numbers and expression levels of essential genes (4CL and CHS) in the (2S)-naringenin metabolic pathway by qPCR. Higher copy numbers of the (2S)-naringenin metabolic pathway genes were associated with greater 4CL and CHS transcription, and the efficiency of naringenin production was higher. Therefore, multi-copy integration of genes in the (2S)-naringenin metabolic pathway was imperative in rewiring p-coumaric acid flux to enhance flavonoid production.

The human rhinovirus 14 3C protease (HRV3C protease), in fusion with glutathione S-transferase also referred to as PreScission™ protease, is a cysteine protease of particular interest for affinity tag removal from fusion proteins due to its stringent recognition sequence specificity (LEVLFQ/GX) and superior activity at low temperature. Here we report the expression, purification and use of a fusion construct of HRV3C protease, NT*-HRV3CP, that affords high expression yield in E. coli (over 300 mg/L cell culture), facile single-step purification, high solubility (>10 mg/mL) and excellent storage properties. NT*-HRV3CP cleaves affinity tags at 4 °C in minutes, making it an attractive tool for the production of recombinant proteins for biotechnological, industrial and pharmaceutical applications.

Astaxanthin shows multiple biological activities, but it is usually linked to different fatty acids and exists in the form of esters. The complexity of astaxanthin esters limits their application in the preparation of sophisticated drugs. Herein, a novel lipase from Streptomyces bacillaris that could hydrolyze astaxanthin esters, named OUC-Sb-lip12, was expressed in Bacillus subtilis. The active site of OUC-Sb-lip12 is probably composed of a dyad of Ser48 and His254, instead of a typical catalytic triad. The lipase was identified to be a GDSL hydrolase, and it showed highest activity at 45 °C and pH 9.0 (glycine-NaOH buffer). OUC-Sb-lip12 showed a good stability at its optimum temperature or a higher temperature, retaining 88.4% and 80.6% of its activity after incubating for 36 h at 45 °C and 55 °C, respectively. OUC-Sb-lip12 could effectively hydrolyze astaxanthin esters in Haematococcus pluvialis oil, generating free astaxanthin. Under the optimum conditions, 96.29% astaxanthin esters were hydrolyzed in 12 h. In addition, B.subtilis is a GRAS model strain and it could efficiently secrete lipase in 9 h, making the lipase potential for scale production of free astaxanthin, which could be further used in the preparation of specific astaxanthin esters with specific functions.

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (FOL), is a worldwide tomato disease. Although Fusarium wilt management remains unsuccessful, enhancing host FOL resistance using magnesium oxide to activate plant immunity may enable effective control. We demonstrated that MgO-pretreatment of roots induced FOL resistance in susceptible tomato plants. Resistance was not induced in tomato mutants deficient in the jasmonic acid (JA) signaling pathway, whereas the opposite trend was observed in mutants deficient in the salicylic acid and ethylene signaling pathways, suggesting that JA signaling activation is essential for MgO-induced FOL immunity. Quantitative real-time polymerase chain reaction analysis of MgO-pretreated tomato plants, and challenge-inoculated with FOL, revealed that MYELOCYTOMATOSIS ONCOGENE HOMOLOG 2 (MYC2), the master regulator of JA signaling, as well as MYC2-targeted transcription factors that directly regulate the JA-induced transcription of late defense genes and their downstream wound-responsive genes were preferentially upregulated in both roots and stems. Moreover, in MgO-pretreated tomato plants challenge-inoculated with FOL, the late wound-responsive THREONINE DEAMINASE 2 (TD) gene was expressed earlier than its upstream genes, including MYC2, suggesting that a primed state for defense was established in MgO-pretreated plants. We conclude that MgO is a promising agent for the control of Fusarium wilt.

As the world population increases and wild caught fisheries decline, aquaculture offers a sustainable solution addressing this global challenge. However, disease management remains difficult. With limited options, there is a need for innovative solutions. The cytokine interleukin-22 (IL-22) has emerged as a possible therapeutic target for fish and has been correlated with protection under pathogen challenge. Plant-based production systems have the potential to effectively manufacture and bring unique efficacy-enhancing features to the aquaculture industry; namely, the advantages of low cost for this commodity market, ready scalability, and reduced environmental impact. Catfish IL-22 produced at significant yield and purity highlights the use of plants as a promising production platform for therapeutic proteins with utility to the aquaculture industry. Purified cfIL-22 shows similar in vitro bioactivity to its mammalian homolog that include increased proliferation of catfish cells highlighting the tissue preservation capabilities associated with this protein. Recombinant cfIL-22 also upregulated expression of genes encoding a tissue repair protein, fibronectin, an antimicrobial peptide, Natural killer lysin-1, and a common innate immune protein, interferon. These findings support plant-made recombinant catfish interleukin-22 as a potential therapeutic for the aquaculture industry and further analysis of this protein for promoting animal health.

Currently, stable Chinese hamster ovary cell lines producing therapeutic, recombinant proteins are established either by antibiotic and/or metabolic selection. Here, we report a novel technology, PTSelect™ that utilizes an siRNA cloned upstream of the gene of interest (GOI) that is processed to produce functional PTSelect™-siRNAs, which enable cell enrichment. Cells with stably integrated GOI are selected and separated from cells without GOI by transfecting CD4/siRNA mRNA regulated by PTSelect™-siRNAs and exploiting the variable expression of CD4 on the cell surface. This study describes the PTSelect™ principle and compares the productivity, doubling time and stability of clones developed by PTSelect™ with conventionally developed clones. PTSelect™ rapidly established a pool population with comparable stability and productivity to pools generated by traditional methods and can further be used to easily monitor productivity changes due to clonal drift, identifying individual cells with reduced productivity.

Ruta chalepensis L., most commonly known as 'fringed rue,' is an excellent and valuable bioactive plant that produces a range of complex flavonoids, of which rutin is the major compound present in this plant of great pharmaceutical and medicinal significance. The present study is a pioneering attempt to examine the changes in the transcriptomic landscape of leaf, stem, and root tissues and correlate this with rutin quantity in each tissue in order to identify the candidate genes responsible for rutin biosynthesis and to increase genomic resources in fringed rue. Comparative transcriptome sequencing of leaves, stems and roots were performed using the NovaSeq 6000 platform. The de novo transcriptome assembly generated 254,685 transcripts representing 154,018 genes with GC content of 42.60 % and N50 of 2280 bp. Searching assembled transcripts against UniRef90 and SwissProt databases annotated 79.7 % of them as protein coding. The leaf tissues had the highest rutin content followed by stems and roots. Several differentially expressed genes and transcripts relating to rutin biosynthesis were identified in leaves comparing with roots or stems comparing with roots. All the genes known to be involved in rutin biosynthesis showed up-regulation in leaves as compared with roots. These results were confirmed by gene ontology (GO) and pathway enrichment analyses. Up-regulated genes in leaves as compared with roots enriched GO terms with relation to rutin biosynthesis e.g. action of flavonol synthase, biosynthetic mechanism of malonyl-CoA, and action of monooxygenase. Phylogenetic analysis of the rhamnosyltransferase (RT) gene showed that it was highly homologues with RT sequence from Citrus species and all were located in the same clade. This transcriptomic dataset will serve as an important public resource for future genomics and transcriptomic studies in R. chalepensis and will act as a benchmark for the identification and genetic modification of genes involved in the biosynthesis of secondary metabolites.

(S)-2-chlorophenylglycine methyl ester ((S)-1) is a key chiral building block of clopidogrel, which is a widely administered antiaggregatory and antithrombotic drug. Herein, Protease 6SD was covalently immobilized on multi-walled carbon nanotubes (MWCNT), and the as-prepared immobilizate P-6SD@NH2-MWCNT was applied in the enantioselective resolution of (R,S)-1 to yield (S)-1. In order to overcome the poor solubility of (R,S)-1 in aqueous solution, a novel triphasic reaction system constituting P-6SD@NH2-MWCNT, aqueous phase and methyl tert-butyl ether (MTBE) as the organic phase was constructed, which simultaneously improved the substrate solubility and the immobilizate recyclability. Under the optimized reaction conditions, P-6SD@NH2-MWCNT catalyzed 10 mM (R,S)-1 for 2 h, yielding optically pure (S)-1 (>99.0 % ees) with 70.74 % conversion of the (R,S)-1. Moreover, P-6SD@NH2-MWCNT can be reused for 15 batches, displaying an exquisite recycling performance. It is for the first time that enantiomerically pure (S)-1 was successfully synthesized by protease-catalyzed one-step resolution.

Although multicopper oxidase from the hyperthermophilic archaeon Pyrobaculum aerophilum (McoP) can be particularly useful in biotechnological applications, e.g., as a specific catalyst at the biocathode of biofuel cells (BFCs), owing to its high stability against extremely high temperatures and across a wide range of pH values, this application potential remains limited due to the enzyme's low catalytic activity. A directed evolution strategy was conducted to improve McoP catalytic activity, and the No. 571 mutant containing four amino acid substitutions was identified, with specific activity approximately 9-fold higher than that of the wild type enzyme. Among the substitutions, the single amino acid mutant F290I was essential in enhancing catalytic activity, with a specific activity approximately 12-fold higher than that of the wild type enzyme. F290I thermostability and pH stability were notably comparable with values obtained for the wild type. Crystal structure analysis suggested that the F290I mutant increased loop flexibility near the T1 Cu center, and affected electron transfer between the enzyme and substrate. Additionally, electric current density of the F290I mutant-immobilized electrode was 7-fold higher than that of the wild type-immobilized one. These results indicated that F290I mutant was a superior catalyst with potential in practical biotechnological applications.

In this paper, a novel procedure for the immobilization and stabilization of enzymes is proposed: the multipoint covalent attachment of bi-molecular enzyme aggregates. This immobilization protocol allows the "capture" and fixation of the enzyme aggregate on the support surface. In addition to stabilization by multipoint attachment, enzyme aggregation promotes very interesting stabilizing effects. In the presence of low concentrations of polyethylene glycol (30 %) the dimeric amine oxidase from Pisum sativum forms soluble bi-molecular aggregates. Enzyme aggregates were analyzed by Dynamic Light Scattering and by full chemical loading of a mesoporous support (10 % agarose gels activated with glyoxyl groups). The soluble aggregate was immobilized by multipoint attachment on glyoxyl- agarose at pH 8.5 though the four amino termini of the two dimeric molecules (Lys residues are not reactive at this pH). The immobilized aggregated structure cannot undergo any movement (translational or rotational) after multipoint attachment and the aggregate is "fixed" on the support surface even after the removal of PEG. The immobilized aggregate was further incubated at pH 10 in order to allow the Lys residues to react with the glyoxyl groups on the support. Enzyme aggregation has an important effect on enzyme stabilization: the aggregated derivative was 40 fold more stable than a similar derivative of the isolated enzyme and 200 fold more than native enzymes in experiments of thermal inactivation.

The compound cis-1,2-dihydrocatechol (DHC) is highly valuable since it finds wide application in the production of fine chemicals and bioactive compounds with medical relevance. The biotechnological process to generate DHC involves a dearomatizing dihydroxylation reaction catalyzed by toluene dioxygenase (TDO) from P. putida F1, employing benzene as substrate. We aimed to enhance the biotechnological E. coli BW25113 platform for DHC production by identifying the key operational parameters positively influencing the final isolated yield. Thereby, we observed an unreported downstream reaction, generating catechol from DHC, affecting, in a negative manner, the final titer for the product. Expression temperature for the TDO-system showed to have the highest influence in terms of final isolated yield. A KEIO-collection-based screening approach highlighted glycerol dehydrogenase (GldA) as the main responsible enzyme for the undesired reaction. We transferred the TDO-system to E. coli BW25113 ΔgldA and applied the enhanced operational set-up on it. This enhanced platform enabled the production of 1.41 g L-1 DHC in isolated yield, which represents a two-fold increase compared with the starting working conditions. To our knowledge, this is the highest DHC production accomplished in recombinant E. coli at semi-preparative scale, providing a robust and accessible biotechnological platform for DHC synthesis.

Bioavailable glutamine (Gln) is critical for metabolism, intestinal health, immune function, and cell signaling. Routine measurement of serum Gln concentrations could facilitate improved diagnosis and treatment of severe infections, anorexia nervosa, chronic kidney disease, diabetes, and cancer. Current methods for quantifying tissue Gln concentrations rely mainly on HPLC, which requires extensive sample preparation and expensive equipment. Consequently, patient Gln levels may be clinically underutilized. Cell-free protein synthesis (CFPS) is an emerging sensing platform with promising clinical applications, including detection of hormones, amino acids, nucleic acids, and other biomarkers. In this work, in vitro E. coli amino acid metabolism is engineered with methionine sulfoximine to inhibit glutamine synthetase and create a CFPS Gln sensor. The sensor features a strong signal-to-noise ratio and a detection range ideally suited to physiological Gln concentrations. Furthermore, it quantifies Gln concentration in the presence of human serum. This work demonstrates that CFPS reactions which harness the metabolic power of E. coli lysate may be engineered to detect clinically relevant analytes in human samples. This approach could lead to transformative point-of-care diagnostics and improved treatment regimens for a variety of diseases including cancer, diabetes, anorexia nervosa, chronic kidney disease, and severe infections.

Conjugation of protein therapeutics with polymers like polyethylene glycol (PEG) has been shown to increase their therapeutic efficiency. However, manufacturing of PEGylated drugs requires an additional, carefully controlled reaction step after purifying the protein, followed by further purification of over- and under-PEGylated variants. In this work, we have used a combined spectroscopic and statistical approach for monitoring and control of the PEGylation reaction for G-CSF using near infrared spectroscopy (NIRS). An online NIRS probe deployed in the reaction vessel has been used to track conversion of G-CSF into monoPEGylated and multiPEGylated forms using calibrated partial least squares regression models on the NIRS spectra which are collected in real time every 3 s. A pH probe integrated with a peristaltic pump facilitates automated quenching of the reaction at the targeted time. The NIRS spectra have also been used to build a batch evolution model for the reaction from end-to-end, including the addition of the reactants to the reaction vessel, the progress of the reaction for 70 min, and the final quenching with Tris base. Online spectra are compared against the statistical process control charts of the batch evolution model in real time to detect deviations as soon as they occur. The system was demonstrated for four common deviations in the PEGylation process, namely: delayed quenching time, wrong concentration of reducing agent added, wrong PEG to G-CSF ratio, and wrong sequence of addition of reactants. The system was able to identify all four deviations in real time and alert the operator to take control actions. The PAT approach suggested here embraces the quality by design framework and can be generalized for manufacturing scale monitoring and control of different biotechnology reactions with spectroscopic signatures.

Gamma-aminobutyric acid (GABA) is a newly recognized signaling molecule participating in physiological processes, growth, and development of plants under optimal and stressful environments. In the present reported research, we investigated the role of GABA in imparting salt stress tolerance in wheat (Triticum aestivum L.). Exposure of wheat plants to 100 mM NaCl resulted in increased oxidative stress, glucose content, nitric oxide (NO) production together with reduced growth and photosynthetic traits of plants. Contrarily, GABA application improved nitrogen (N) metabolism, sulfur (S) assimilation, ion homeostasis, growth and photosynthesis under salt stress. Additionally, GABA mitigated oxidative stress induced by salt stress with the increased ascorbate-glutathione cycle and proline metabolism. The study with NO inhibitor, c-PTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide] in GABA experiment suggested that the impact of GABA on improvement of growth and photosynthesis under salt stress was mediated by NO and influenced N and S assimilation and antioxidant systems. The results suggested that the GABA has a significant potential in reversing the salt stress response in wheat plants, and GABA-mediated signals are manifested through NO.

Leuconostoc citreum is a heterofermentative lactic acid bacterium frequently found in the various fermented foods. L. citreum EFEL2700 isolated from Korean kimchi has been used as a host strain for biotechnological applications. For the use as a food-grade host to over-produce food ingredients or enzymes, strong endogenous promoters guarantying high expression levels of target genes are necessary. In this study, transcriptomic analysis of L. citreum EFEL2700 was performed using RNA-Seq and three promoters of the most highly expressed genes were selected: glyceraldehyde 3-phosphate dehydrogenase (G3PD), 6-phosphogluconate dehydrogenase (6PGD), and phosphoketolase (PPK). Thereafter, they were used as promoters to express β-galactosidase gene from Lactobacillus plantarum WCFS1 in L. citreum EFEL2700 and the levels were compared with the control promoter P710 from L. mesenteroides ATCC 8293. As results, the β-galactosidase activities of the transformants were 2.73, 0.27, 37.43, and 9.25 units/mg under the P710, G3PD, 6PGD, and PPK promoters, respectively. The expression level of endogenous promoter 6PGD was superior to the heterologous P710 promoter previously used in a Leuconostoc-Escherichia coli shuttle vector. The 6PGD developed in this study can be used as the most suitable promoter for β-galactosidase expression in L. citreum EFEL2700.

In this study, mix-culture fermentation (MF) with M. anka, S. cerevisiae and B. subtilis to regulate phenolic release in corn seeds was investigated. Results showed the adding strategy of S. cerevisiae and B. subtilis significantly (p < 0.05) influenced the total phenolic content (TPC) of corn seeds, and the highest TPC in MF was 22.56-fold rise than that of unfermented control. Moreover, the phenolic compounds in corn seeds were shifted after MF. SDS-PAGE revealed some enzymes produced by microorganisms were regulated, and the α-amylase and FPase activities were changed to improvement in MF. SEM micrographs of corn seeds in MF varied markedly with the cell walls structure destroyed, so as to release of the phenolic fractions in corn seeds. Based on the phenolic mobilization, DPPH and ABTS+ radical scavenging abilities of phenolic fractions from corn seeds were improved in MF. This paper provides a prospect fermentation process for improving the phenolic release in corn cereals.

Lipid hyperaccumulation in oleaginous microorganisms is generally induced by nitrogen limitation, while oxygen supply can influence biomass growth and cell metabolism. Although strategies based on nitrogen limitation or oxygen control have been extensively explored and applied in various oleaginous microorganisms, the role of oxygen supply in nitrogen limitation induced lipid hyperaccumulation still remains unclear. Here, we systematically surveyed the effects of oxygen supply on the oleaginous fungus M. alpina cultured in nitrogen limited conditions through integration of physiochemical parameters and metabolomics analysis. Our results indicated that a high oxygen supply promoted carbon/nitrogen consumption and was used for rapid biomass synthesis, while either high or low oxygen supply conditions were adverse to lipid and ARA accumulation. Different oxygen supply level significantly affected the balance between fermentation for lipid synthesis and respiration for energy generation. Under nitrogen limitation, a suitable oxygen supply promoted the recycling of preformed nitrogen and increased the redirection of carbon towards fatty acid synthesis through the hub centred around glutamic acid coupled to the intermediate metabolism of carbon in the TCA cycle, while a high oxygen supply favored the respiration process and enhanced the degradation of LC-PUFAs, rather than fermentation for fatty acid synthesis. This system-level insight reveals the underlying metabolic mechanism of oxygen control in nitrogen limitation induced lipid accumulation, and provides theoretical support for the integration of oxygen control with nutrient supply for efficient microbial oil production.

Characterization of stress-responsive genes is important to understand the genomics perspective of stress tolerance. In this purview, several gene-families are being identified and characterized in the model and non-model plant species, which has greatly enhanced the knowledge of molecular intricacies associated with stress tolerance. One such gene family is the GRAM-domain containing which have been found to be upregulated in response to plant growth-promoting rhizobacteria (PGPR) treatment followed by salinity stress. Thus, we aimed at understanding the involvement of GRAM domain-containing proteins in abiotic stress response under the influence of rhizobacteria in Arabidopsis thaliana. The study identified fourteen AtGRAM genes in A. thaliana. Further, comprehensive analyses of domain family including phylogenetic studies, domain architecture, gene structure and genomic composition analysis, promoter analysis, homology modelling, and duplication and divergence rates estimation was performed. RNA-Seq derived expression profiling of AtGRAM genes using GENVESTIGATOR in different stresses, developmental stages and hormonal treatments was performed, followed by qRT-PCR analysis under abiotic stresses in response to PGPR. Altogether, the study provided insights into the structure, organization, and evolutionary properties of AtGRAM gene family. Modulation in expression pattern in response to stresses influenced by PGPR-treatment suggests its multifaceted role in cross-talk among abiotic stresses and phytohormones. Further functional characterization of the selected candidate genes would enable understanding of the precise roles of GRAM-genes underlying stress tolerance.

The photosynthetic acclimation of extremophile Eutrema salsugineum plants to red light (RL) (14 days, 150 μmol photons m-2 s-1, 660 nm) and the expression of the key photoreceptor apoprotein genes, transcription factors (TFs) and associated with phytochrome system MIR (microRNA) genes were studied. RL exposure induced an increase in the content of anthocyanin and total phenolic compounds and the level of Chls was decreased. The photosystem 2 electron transport rate and the number of open reaction centres (qL) were not changed in RL plants, however, the levels of non-photochemical quenching (NPQ) and the regulated quantum yield of non-photochemical quenching Y(NPQ) were significantly higher in the RL plants. The rate of CO2 uptake was decreased by almost 1.4-fold but the respiration and transpiration rates, as well as the stomatal conductance were not changed in the RL plants. An increase in the expression of the photoreceptor apoprotein genes PHYA, PHYB and PHYC, the TF genes PIF4, PIF5 and miR395, miR408, miR165 and decreases in the levels of the transcripts of the TF gene HY5 and miR171, miR157, and miR827 were detected. The acclimation effect of photosynthetic apparatus to RL was accompanied by an increase of pigment content such as total phenolic compounds and carotenoids and it is due to the changes in the expression of the apoprotein phytochrome genes PHYA, PHYB, PHYC and phytochrome signalling TFs (PIF4, PIF5 and HY5) as well as MIR genes associated with phytochrome system.

With the advent of rapid evolution of oomycete pathogen lineages, the need for sustainable agriculture practices has become the need of the hour. The late blight of tomato caused by Phytopthora infestans, has recently emerged as one such devastating disease in India that led to huge crop losses. Hence, in the present work seed priming with mycogenic selenium nanoparticles (SeNPs) for elicitation of resistance against tomato late blight disease is investigated. It also aims to understand the defense responses triggered by SeNPs at cellular, biochemical and transcriptomic levels. Enhanced plant growth parameters were observed in bioactive SeNPs-primed tomato plants as compared to control plants. SeNPs-primed and pathogen inoculated plants exhibited a significant protection of 72.9 % against late blight disease. The primed plants also recorded a remarkable accumulation of lignin, callose and hydrogen peroxide that serve as the cellular defense over the control plants. Further, an elevated level of lipoxygenase (LOX), phenylalanine lyase (PAL), β-1,3-glucanase (GLU), superoxide dismutase (SOD) corroborated the biochemical defense in primed plants, which was also reflected in the corresponding transcriptome profiling of the genes encoding the enzymes. Thus, the present study represents an orchestrated correlation between resistance and defense responses incited by SeNPs against tomato late blight disease, which can be used as nano-biostimulant fungicide in protecting tomato plants.

Cytochrome P450scc system performs the first rate-limiting stage of steroidogenesis in mammals. The bovine P450scc system was reconstructed in Saccharomyces cerevisiae, using a foot-and-mouth disease virus 2A peptide (F2A)-based construct, to co-express cytochrome P450scc, adrenodoxin (Adx), and adrenodoxin reductase (AdR). During the translation of the self-processing fusion protein P450scc-F2A-Adx-F2A-AdR, the first and the second linkers are cleaved with different efficiencies (96 % and 11 %, respectively), resulting in the unbalanced expression of individual proteins. The low cleavage efficiency and the relative Adx and AdR protein levels were increased through replacing the second F2A peptide with different sequences and changing the order of Adx and AdR. The P450scc, AdR, and Adx sequences located upstream of the F2A affected F2A processing, to various degrees. Moreover, using molecular dynamics (MD) simulations, we showed that the 2A peptide fused to the C-terminus of Adx formed the steric hindrance during enzymatic complex formation, resulting in the reduction of catalytic activity. Thus, the functional activity of the reconstructed P450scc system was determined not only by the efficiency of 2A peptides but also by the overall sequence of the expressed 2A-polyprotein. Our results can be applied to the development of 2A-based co-translation strategies, to produce other multicomponent protein systems.

Development of new non-toxic antioxidants with diverse hydrophobic properties is important due to growing concerns about the toxicity of artificial oil-soluble antioxidants, the comparatively low effectiveness of natural options, and the complex role hydrophobicity plays in antioxidant effectiveness. Using caffeic acid, a naturally occurring phenolic acid with potent antioxidant activity, a range of glyceryl caffeate esters (decanoate and palmitate) were prepared using lipase-catalysed esterification reactions. Glyceryl-1-caffeate (GC) was prepared from ethyl caffeate and glycerol (acting as both the solvent and the substrate), catalysed by immobilised Candida Antarctica lipase B (Novozym-435) at 80 °C under vacuum. Esterification of GC with decanoic acid using immobilised Thermomyces lanuginosus lipase (TLIM) or Novozym-435 was found to be selective towards mono-acylated or di-acylated products, respectively. The reaction was performed in an unconventional solvent, propylene carbonate (PC), which has many of the attributes of a green solvent. Product conversions in PC were comparable to the best performing conventional solvents. In contrast to conventional volatile solvents, the low volatility of PC allowed the reaction to be performed under vacuum, without the need for molecular sieves for removal of water produced during the reaction. Diisopropyl ether was effective at extracting the more lipophilic products from PC. Both the lipase (Novozym-435) and PC were reused four times with only a small loss in conversion efficiency. Glyceryl caffeate esters performed much better than α-tocopherol at protecting bulk tuna oil from oxidation (analysed using Rancimat). A comparison of glyceryl caffeate esters (decanoate/palmitate and mono-/di-acylated) showed that their antioxidant effectiveness in bulk tuna oil was not affected by chain-length, but compounds containing only one fatty ester were slightly more effective than those containing two fatty esters.

A Tetraselmis sp. was selected for its antioxidant activity owing to its high lipid peroxidation inhibition capacity. With the aim to monitor culture conditions to improve antioxidant activity, effects of CO2-induced acidification on Tetraselmis growth, elemental composition, photosynthetic parameters and antioxidant activity were determined. Two pH values were tested (6.5 and 8.5) in batch and continuous cultures in photobioreactors. Acidification enhanced cell growth under both culture methods. However, the microalgae physiological state was healthier at pH 8.5 than at pH 6.5. Indeed, photosynthetic parameters measured with pulse amplitude modulated (PAM) fluorometry showed a decrease in the photosystem II (PSII) efficiency at pH 6.5 in batch culture. Yet, with the exception of the PSII recovering capacity, photosynthetic parameters were similar in continuous culture at both pH. These results suggest that lowering pH through CO2-induced acidification may induce a lower conversion of light to chemical energy especially when coupled with N-limitation and/or under un-balanced culture conditions. The highest antioxidant activity was measured in continuous culture at pH 6.5 with an IC50 of 3.44 ± 0.6 μg mL-1, which is close to the IC50 of reference compounds (trolox and α-tocopherol). In addition, the principal component analysis revealed a strong link between the antioxidant activity and the culture method, the photophysiological state and the nitrogen cell quota and C:N ratio of Tetraselmis sp.. These results highlight Tetraselmis sp. as a species of interest for natural antioxidant production and the potential of PAM fluorometry to monitor culture for production of biomass with a high antioxidant activity.